We have continued our studies to elucidate the molecular basis of heritable disorders of Connective tissue disease and to apply this information to the treatment of these disorders. Using the antisense riboprobe system, we have detected mismatches in the type I collagen mRNA of three cases of osteogenesis imperfecta (OI) and are now engaged in isolating the mutant alleles for sequencing: a case of lethal OI with a mismatch in the COOH end of alpha 2(I), type III OI with a mismatch in the middle of alpha I(I), and type IV OI with mismatches in the 5'-end of alpha I(I) and the 3'-end of alpha 2(I). We are extending our ability to detect mismatches by the implementation of chemical cleavage methodology and by using both sense and antisense riboprobes to screen both strands of PCRamplified cDNA. We are applying these techniques especially to those cases in which there is evidence of mosaicism, compound heterozygosity or variability of expression. We have begun to develop a riboprobe system and protein isolation techniques for type III collagen. The combination of systems to detect point mutations in types I and III collagen is aimed at molecular studies of patients with various forms of Ehlers-Danlos (ED) syndrome. We have analyzed the protein synthesized by cultured fibroblasts in five cases of ED and have detected electrophoretic abnormalities in the type I collagen in three cases; the mRNA of these cases is now being screened by the riboprobe technology. In clinical protocols, we have continued (1) our investigation of the growth deficiency in OI and the responsiveness of OI bone to growth stimulation, and (2) the physical rehabilitation and bracing protocol for children with moderately severe OI. We have also initiated a collaborative investigation of the dynamics of skeletal calcium in OI children.